Vanilla is the world's most popular flavor compound and used in numerous commercial products including many foods. Natural vanilla is obtained from the pods of the epiphytic climbing orchids Vanilla planifolia Andrews and Vanilla tahitensis belonging to the genus Vanilla included in the order Orchidales within the monocotyledonous plants. Vanillin (3-methoxy-4-hydroxybenzaldehyde) is the main flavor compound in the vanilla extract obtained from fermented vanilla pods. In high concentrations, vanillin is toxic to living cells. In the pod it accumulates as the non-toxic phytoanticipin vanillin glucoside, which upon tissue damage is converted into the active defense compound. During fermentation and curing of the vanilla pods for use as flavor ingredient, a major part of the vanillin glucoside is hydrolyzed to provide free vanillin. The estimated total world market for vanillin is 10500 tons per annum. Production of natural vanillin from the vanilla pods is a laborious, slow and costly process. Production of 1 kilogram of vanillin requires approximately 500 kilograms of vanilla pods, corresponding to the pollination of approximately 40,000 flowers. Nowadays, only 3% of the global vanillin production is derived from vanilla pods. The vast majority is produced synthetically from different fossil hydrocarbons like eugenol or by acid hydrolysis of lignin. Biotechnological production of vanillin in microorganisms using heterologous expression of the genes encoding the pathway in vanilla orchids has not been achieved because none of the genes have been identified. Instead, vanillin has been produced in yeast, fungi, bacteria and in in vitro cell cultures by expressing genes from other organisms which encode enzymes that in combination would form vanillin from exogenously added substrates structurally related to vanillin.
Phenylalanine-derived volatiles are categorized as C6-C1, C6-C2, C6-C3 compounds based on their carbon skeleton. Both vanillin glucoside and p-hydroxybenzaldehyde glucoside, the two most abundant compounds found in mature vanilla pods, are C6-C1 compounds.
The two major aroma compounds in natural vanilla are p-hydroxybenzaldehyde and vanillin. Because p-hydroxybenzaldehyde contains some of the same structural elements of vanillin, it has been thought of as a precursor for vanillin. The pathway from vanillin has been postulated to constitute a metabolic grid of metabolites all derived from phenylalanine. US2003/0070188 describes methods for possible p-hydroxybenzaldehyde production in embryogenic cell cultures from Vanilla planifolia. One method described in US2003/0070188 takes advantage of a 4-hydroxy-benzaldehyde synthase (4-HBS), which is described as being capable of catalyzing chain shortening of p-coumaric acid to yield p-hydroxybenzaldehyde. The document further describes expression of 4-HBS in creeping bentgrass, however no information regarding the outcome of such expression is provided. It is also described that no 4-hydroxybenzaldehyde could be detected in yeast expressing 4-HBS.
Podstolski et al., 2002 describes that 4-hydroxy-benzaldehyde synthase (4-HBS) converts 4-coumaric acid non-oxidatively to 4-hydroxybenzaldehyde in the presence of a thiol reagent but with no co-factor requirement.